JP2003088398A - Method for measuring specimen by light emission and device therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a small, lightweight and inexpensive device for measuring a specimen. SOLUTION: This device 1 for measuring a specimen by light emission comprises a chamber 9 for receiving an examination tool, a low sensitive light- receiving element 5 for receiving light emitted from an examination tool placed in the receiving chamber 9, and a processor 46 for electrically processing signals from the low sensitive light-receiving element 5 such as magnesium photodiode, gallium arsenic photodiode, gallium arsenic phosphorus photodiode, or iridium gallium arsenic photodiode. The contamination of the specimen is made to emit light with a luminescent reagent containing an ATP-reproducing enzyme such as PPDK, and the emission is measured with the low sensitive light- receiving element 5.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and an apparatus for measuring adenosine phosphates by allowing a luminescent reagent containing an ATP regenerating enzyme or a luminescent reagent not containing an ATP to act on a sample.

[0002]

2. Description of the Related Art Adenosine triphosphate (hereinafter referred to as ATP) is used for detecting stains derived from bacteria or foods by using a reagent containing luciferase and luciferin.
It is known that it can be measured by a luminescence reaction using (). However, the output of this light is extremely small, and in the past, in order to obtain a measurable output, a photomultiplier tube (photomultiplier) was used in the photodetector. However, since the photomultiplier tube requires a high voltage, it needs a voltage conversion device and measures for safety, and thus has a drawback that the device is large and expensive.

Further, a photodetector using an avalanche photodiode (Tokuhoku 4-502403) is also known. This device requires a temperature stabilization system, since avalanche photodiodes typically need to maintain a working temperature of 0-5 ° C. Further, this device has a Peltier effect type heat pump mounted adjacent to the avalanche photodiode, and receives the current supply from the temperature control circuit to cool the avalanche photodiode. Therefore, the device becomes complicated,
It had the drawback of being expensive.

[0004]

The present inventor has found that the sensitivity is not so high, but a light receiving element such as a germanium (Ge) photodiode or a gallium arsenide (GaAs) photodiode, which does not require a high-voltage, high-current power source, Gallium arsenide phosphorus (GaAsP) photodiode, iridium gallium arsenide (InGaAs) photodiode, phototransistor, cadmium sulfide (CdS) photoconductive cell (visible light conductive element), PIN photodiode, photo IC, Charge Couple
We focused on d Devices (CCD elements), MOS elements, etc. (hereinafter collectively referred to as low sensitivity light receiving elements). On the other hand, the inventor of the present application previously applied for "a bioluminescence reagent, a method for quantifying adenosine phosphate using the reagent, and a method for quantifying a substance relating to an ATP conversion reaction system using the reagent" (Japanese Patent Laid-Open No. 9-234099). did. The present invention provides at least pyruvate orthophosphate dikinase (hereinafter referred to as PPDK, Pyruvate).
This is a method in which a bioluminescence reagent is constituted by a reagent containing an ATP regenerating enzyme such as orthophospate dikinase, EC 2.7.9.1), and a high luminescence amount can be obtained even with a trace amount of adenosine phosphates. Therefore, the inventor of the present invention further diligently researched,
Adenosine phosphates, that is, stains can be detected with a simple device by detecting adenosine phosphates with a luminescent reagent containing ATP regenerating enzyme such as PPDK and measuring the amount of emitted light with a low-sensitivity light receiving element. Based on this knowledge, the present invention was completed.

[0005]

[Means for Solving the Problems] That is, the present invention is characterized in that adenosine phosphates are caused to emit light by a luminescent reagent containing an ATP regenerating enzyme, and the amount of emitted light is measured by a low-sensitivity light receiving element. A method for measuring a sample, a chamber for storing an inspection instrument, a low-sensitivity light receiving element for receiving light emitted by the inspection instrument placed in the chamber,
And a device for measuring a sample by light emission, which comprises an arithmetic unit for electrically processing a signal from the low-sensitivity light receiving element.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a storage chamber for storing an inspection instrument that collects dirt and emits it, a low-sensitivity light receiving element for receiving the emitted light, and the received light are measured. There is provided an apparatus for measuring a sample, which includes an arithmetic unit for the above, and further includes an operation panel or a display panel. The low-sensitivity light receiving element is not the same as the photomultiplier tube,
It is a semiconductor device that outputs a measurable electrical signal in response to low-level light. Moreover, a high-voltage or high-current power source is not required for the circuit portion of the low-sensitivity light receiving element, and a battery can be used as the power source. Further, the low-sensitivity light receiving element has less change in performance with respect to temperature changes than other general diodes such as avalanche photodiodes, and does not require a temperature stabilizer. Further, the low-sensitivity light receiving element is more robust than the photomultiplier tube and is not adversely affected even when exposed to strong light. Therefore,
The cleanliness measuring device according to the present invention can be significantly smaller, lighter, and less expensive than conventional devices.

The luminescent reagent used in the present invention contains an ATP regenerating enzyme such as PPDK and can be used by adding it to the luciferin / luciferase luminescent reagent.
A luminescent reagent containing an ATP regenerating enzyme emits light by reacting not only with ATP but also with adenosine diphosphate (hereinafter referred to as ADP), adenosine monophosphate (hereinafter referred to as AMP), and ribonucleic acid (hereinafter referred to as RNA). And exhibits a stable and high level of light emission. Therefore, it is possible to compensate for the defect that the sensitivity of the low-sensitivity light receiving element is slightly lower than that of the photomultiplier tube. Needless to say, a usual luciferin luciferase luminescent reagent containing no ATP regenerating enzyme, a luminescent reagent based on chemiluminescence, or a fluorescent reagent can also be used. The dirt referred to in the present invention means ATP, ADP, AM
It contains adenosine phosphates such as P and RNA, and preferably, a stain inspection in the cleanliness inspection can be mentioned.

[0008] The ATP regenerating enzyme referred to in the present invention is AM
Any enzyme that catalyzes the reaction of producing ATP from P may be used. It also includes the case where a reaction of producing ATP from AMP is performed by combining enzymes. For example, Phosphoenolpyruvate Synt
hetase, EC 2.7.9.2). In addition, ATP may be produced from AMP by using adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) in combination.
Also, as described in JP 2001-211900 A, ATP is produced from AMP by using a combination of polyphosphate kinase and adenylate kinase (EC 2.7.4.3). good. In addition, AMP-PolyP phosphotransferase (AMP-Polyph
osphate phosphotransferase) and polyphosphate kinase (P
ATP may be generated from AMP using a combination of olyphosphate kinase). Also, Applied and Enviro
nmental Microbiology, May 2000, P2045-P2051 as described in polyphosphate: AMP phosphotransferase.
And Adeny rate kinase may be combined to generate ATP. Also, Journal of Biochemistry 128, P1079-P108
5 ADP-Dependent (AMP-Forming) Gl described in 2000
ucokinase was used to generate AMP from ADP, and then pyruvate orthophosphate dikinase was used.
ATP may be generated from AMP. Preferably PPD
K can be used.

The PPDK used in the present invention is an enzyme which acts on AMP, phosphoenolpyruvate and pyrophosphate in the presence of a metal ion such as magnesium to catalyze the reaction to produce ATP, pyruvate and phosphate. Its physicochemical properties and production method are already known and readily available (see Japanese Patent Laid-Open No. 9-234099). Examples of the luminescent reagent containing the ATP regenerating enzyme include luminescent reagent containing luciferin, luciferase and metal salt, and PP.
DK is added with phosphoenolpyruvate,
A better effect can be obtained by adding pyrophosphoric acid at the same time. By using this, it is possible to simultaneously measure AMP in addition to ATP, which is an index component of dirt, and it is possible to detect a small quantity of dirt with high sensitivity. When an enzyme that catalyzes a reaction for producing ATP from ADP and / or an RNA degrading enzyme is further used in combination with the above-mentioned ATP regenerating enzyme-containing luminescent reagent, in addition to ATP and AMP, ADP
Also, RNA and RNA can be measured at the same time to detect even less stains.

In the practice of the present invention, the stain on the surface to be tested is wiped with a cotton swab and the cotton swab is used to extract adenosine phosphates such as ATP, ADP, AMP and RNA from stains such as bacteria. Immerse it in an extraction reagent containing a surfactant or the like. The sample solution thus obtained is mixed with a luminescent reagent containing an ATP regenerating enzyme such as PPDK, and the amount of emitted luminescence is measured by a sample measuring device, whereby the contamination of the sample can be detected. Preferably, using a swab, an extraction reagent, a cleanliness inspection device in which a luminescent reagent is integrated, that is, a wiping inspection device,
The inspection can be performed more easily. If the device is configured with a wiping test instrument including a luminescent reagent containing an ATP regenerating enzyme such as PPDK, it is possible to provide a small, lightweight, low cost, and highly sensitive cleanliness test method. That is, the surface to be tested is wiped with a cotton swab of a wiping test instrument, immersed in an extraction reagent provided in the instrument, and the obtained sample solution is treated with an ATP regenerating enzyme such as PPDK provided in the instrument. The reaction is carried out with the contained luminescent reagent, and the apparatus for wiping inspection is set in a cleanliness inspection device, and the amount of dirt is measured from the amount of luminescence obtained. All of these devices can be configured to be portable in a carrying case. Therefore, according to the present invention, it is possible to provide a cleanliness inspection kit including a luminescence measuring device, a cotton swab, an extraction reagent, and a wiping inspection instrument in which a luminescence reagent is integrated. The luminescence measuring device described above can also be used for testing various other samples, for example, testing raw materials and final products in the food industry, reagents, water quality, and clinical samples.

The present invention will be specifically described below with reference to the drawings. First, in FIG. 1, reference numeral 1 is a sample measuring device, and a main body 2 thereof has a wiping test instrument 3 for wiping a specimen, that is, a stain and performing a bioluminescence reaction, and a light collecting device for collecting light emission of the wiping test instrument 3. A lens 4, a low-sensitivity light receiving element 5 for converting light from the lens 4 into an electric signal, and an arithmetic unit 46 for processing the electric signal from the light receiving element 5 are housed.

In FIG. 2, 6 is a power supply circuit section 4.
1, an amplifier unit 42, an A / D conversion unit 43, an arithmetic unit 46 including a CPU 44 and a memory 45, a display unit 47, an operation unit 4
8 and an external interface 49. First, the power supply circuit unit 41 is for accepting both a DC power supply and an AC power supply. The amplifier section 42 is for converting the output to a voltage and amplifying the voltage according to the intensity of light emission of the wiping inspection tool 3, that is, the intensity of the electric signal from the low sensitivity light receiving element 5. The voltage signal from the A / D converter 4
It is digitized in 3 and transmitted to the arithmetic unit 46.
The arithmetic unit 46 receives the signal from the low sensitivity light receiving element 5,
This is converted into a numerical value and displayed on the display unit 47, or compared with data of the degree of contamination stored in the memory 45 in advance as necessary, and a countermeasure such as necessity of cleaning is displayed. Reference numeral 50 denotes the amplifier section 42 and the low-sensitivity light receiving element 5
A shield member formed of a metal such as aluminum or iron to cover the outer surface can block noise from the outside.

The operation unit 48 gives an instruction to the CPU 44, and saves measurement data or transmits the data to the printer via the external interface 49 or to a remote place, and further inputs the data to the memory 45. And so on. Further, as shown in FIG. 3, an operation unit 48 and a display unit 47 are provided on the front surface of the main body 2, and these are electrically connected to the arithmetic unit 46 as shown in FIG. 2 described above. The measurement conditions are set or started, and the results are displayed. In FIG. 2, the condenser lens is omitted. The main body 2 is further provided with a storage chamber 9 for the wiping test tool 3, and the tool 3 is placed on the table member 10 and is always positioned at the same position. 11 is a wiping inspection tool 3
With the reflecting mirror arranged on the back surface of the above, more light emission amount can be sent to the low-sensitivity light receiving element 5 via the condenser lens 4.

Next, regarding the wiping test tool 3, this tool 3 is a light emitting reagent such as A such as PPDK.
Any structure may be used as long as the TP regenerating enzyme can be used, and an example thereof is shown in FIG. First, in FIG. 4, the wiping test instrument 3 mainly includes a sample collecting tool 12 and a test tube-shaped luminescent reagent container 13. The sample collecting tool 12 is the sample wiping tool 1
4, an extract liquid container 15, and a main body 16 of the sample collecting tool 12 having a tubular shape. The opening 17 of the luminescent reagent container 13 is closed by a sealing material 18 formed of, for example, aluminum foil, and the luminescent reagent container 13 is sealed.
The sample wiping tool 14 includes a swab 21 including a swab 19 having a rod shape and a cotton part 20 formed and installed in an egg shape at a lower end thereof.
And a holding member 22 for holding the cotton swab 21.

The holding member 22 comprises a lower-diameter portion 23 and an upper-diameter portion 24, and a step portion 25 at the boundary between the small-diameter portion 23 and the large-diameter portion 24 abuts on an upper end portion 26 of the main body 16 and It has a function of always stopping at a fixed position when the wiping tool 14 moves downward in the main body 16. The sample wiping tool 14 is the main body 1
6 is detachably attached to the body 6, and has a function of wiping the surface of the specimen removed from the body 16 with a cotton swab 21 to collect dirt. The extraction liquid container 15 is filled with an extraction liquid 32 for extracting stains attached to the cotton portion 20 of the cotton swab 21 by wiping the sample. The upper and lower ends of the extract liquid container 15 are opened. First, the lower part is sealed with the seal material 27, the extract liquid 32 is injected, and then the upper part is sealed with the seal material 2.
8 is sealed and the extraction liquid 32 is enclosed. The sealing materials 27 and 28 are such that they are easily broken by pressing the cotton swab 21, and are made of, for example, aluminum foil.

The main body 16 of the sample collecting tool 12 is composed of a tubular member whose upper and lower ends are open, and an annular projecting member 29 is provided on the inner wall below, and the projecting member is further provided. The anti-scattering member 30 is connected to the lower part of 29 and extends. Then, by pushing up the luminescent reagent container 13, the sealing material 18 is broken by the acute angle portion 31 of the scattering prevention member 30, and the dirt extracted from the cotton part 20 is dropped and sent out to the luminescent reagent container 13 together with the extraction liquid to emit light or the like. Is configured to detect dirt.

[0017]

First, the sample wiping tool 14 is pulled out from the main body 16, and the surface of the sample is wiped with the cotton part 20 to collect dirt.
Next, after inserting the sample wiper 14 from which dirt has been collected into the main body 16, the luminescence reagent container 13 is pushed up and the sealing material 18 of the container 13 is pressed against the acute angle portion 31 of the scattering prevention member 30 to break the sealing material 18. . Next, when the sample wiping tool 14 is pushed down, first, the sealing material 28 is broken by the cotton swab 21, and the cotton portion 2
0 enters the inside of the extraction liquid container 15. Then, next, the sample wiping tool 14 is pushed down until the stepped portion 25 of the holding member 22 and the upper end portion 26 of the main body 16 come into contact with each other, and the sealing material 27 is moved to the swab 21.
Break by.

Then, the wiping test instrument 3 as a whole is shaken lightly in the vertical direction to drop the extract 32 containing bacteria and dirt into the luminescent reagent container 13 and the luminescent reagent 33.
Upon contact with, this part will glow, for example by a luminescent reaction. Then, the wiping test tool 3 is mounted in the storage chamber 9 of the stain measuring apparatus 1 and the amount of light emission, that is, the amount of stain is measured. As the luminescent reagent 33 in the wiping test device 3 described above, the one containing an ATP regenerating enzyme such as PPDK is used. Although the case of wiping as a sample has been described above, the liquid, solid matter, granular material, etc. to be inspected are put into a test tube containing a luminescent reagent containing an ATP regenerating enzyme such as PPDK, etc. May be measured. Needless to say, the device according to the present invention can measure not only bioluminescence but also chemiluminescence or fluorescence as a sample.

[0019]

EXAMPLES Next, examples will be shown to numerically show the effects of the present invention. Enzyme units are international units.

[0020]

[Example 1] Composition of luminescent reagent containing PPDK         EDTA (ethylenediaminetetraacetate) 1.0 mM         Dithiothreitol 1.0 mM         Ammonium sulfate 3.75 mM         Pyrophosphate 0.3 mM         Phosphoenolpyruvate 2.1 mM         Luciferin 0.75mM         Magnesium sulfate 7.5 mM         BSA (bovine serum albumin) 0.5%         Sucrose 5.0%         Luciferase 2.5mg / ml         PPDK 1.5U / ml         Adenosine phosphate deaminase 0.05U / ml         HEPES (50mM) pH7.8 Composition of extraction reagent         Benzalkonium chloride 0.02%         HEPES (10mM) pH7.8

The luminescent reagent containing the PPDK having the above composition was freeze-dried, and the luminescent reagent container 13 of the wiping test instrument 3 shown in FIG. Yes). On the other hand, the luminescent reagent obtained by lyophilizing the luminescent reagent from the above composition except for PPDK was similarly enclosed in a luminescent reagent container. However, the same extraction reagent was set in the extraction liquid container 15 (without PPDK). As a model of stains derived from food, after dipping the cotton swab 21 of the wiping test device 3 into a commercially available yeast extract, beef extract, malt extract, beer, milk diluted with ultrapure water to each concentration, a swab holder Then, the head of the cotton swab was dipped into the extraction reagent, and the cotton swab holder was further pressed. In the case of fingers, soak the cotton swab 21 in water beforehand, wipe off the palm and fingers with the cotton swab, push the cotton swab holder into the extraction reagent, and then push the cotton swab holder into the extraction reagent. Was sent to the luminescent reagent container 13 containing the freeze-dried luminescent reagent to emit light.

Next, the wiping test tool 3 was set in a sample measuring device 1 using a low-sensitivity light receiving element (GaAsP photodiode), and the amount of light emission was measured. The measured values are shown in relative light emission amount RLU (Relative light Unit), and the results are shown in Table 1. From this Table 1, when the luminescence reagent containing no PPDK is used (no PPDK), only the luminescence amount derived from ATP is obtained, and the luminescence amount is abruptly attenuated and unstable. It was 0 in most cases. On the other hand, in the case of a luminescent reagent containing PPDK (with PPDK), AMP contained in a large amount in food other than ATP
The original luminescence was obtained and the amount of luminescence was stable, so that stains could be measured with high sensitivity. Moreover, since the ratio of ATP to AMP is higher in the case of fingers than in the case of food, it was possible to measure with a relatively high sensitivity even with a luminescent reagent containing no PPDK.

[0023]

[Table 1]

[0024]

[Example 2] In Example 1, the case of the luminescent reagent containing PPDK is PPDK, and from this reagent, PPDK is used.
In the case of the luminescent reagent except only the sample, no PPDK was used. P
200 μl of each with PDK and without PPDK was sampled in a test tube, and 100 μl of each of commercially available orange juice diluted with ultrapure water to 1% was added. This was set in the measuring device 1 of the sample in which the GaAs photodiode was set, and the amount of light emission was measured. The measured value is the relative light emission amount RLU (Relati
ve light Unit). The measured value without PPDK was 124 RLU, whereas the measured value with PPDK was 1356 RLU.

[0025]

EFFECTS OF THE INVENTION Since a luminescence reagent containing an ATP regenerating enzyme such as PPDK is used as the luminescence reagent, the luminescence is strong and stable, and the luminescence can be detected even by a low-sensitivity light receiving element, and the device is small and lightweight. Can be converted.

[Brief description of drawings]

FIG. 1 is a front sectional view of a sample measuring device.

[Fig. 2] Circuit diagram

FIG. 3 is a front view of a sample measuring device.

[Fig. 4] Wiping inspection tool

[Explanation of symbols]

1 Sample measuring device 2 body 3 Wiping inspection instruments 4 condenser lens 5 Low-sensitivity photo detector 6 Measuring circuit 9 storage rooms 10 Table member 11 Reflector 12 Sample collection tools 13 Luminescent reagent container 14 Sample wiping tool 15 Extraction liquid container 46 arithmetic unit 47 display 48 Operation part

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2G054 AA10 AB03 BB01 BB02 BB10                       CA20 EA01 EA02 EA03 FA17                       FA33 JA00 JA09                 4B029 AA07 BB16 FA03 FA09 FA12                 4B063 QA01 QA20 QQ17 QQ20 QQ62                       QQ63 QR07 QR66 QS28 QS39                       QX02

Claims (5)

[Claims] 1. A method for measuring a sample by luminescence, wherein adenosine phosphates are caused to emit light by a luminescence reagent containing an ATP regenerating enzyme, and the amount of luminescence is measured by a low-sensitivity light receiving element. 2. An accommodating chamber for an inspection instrument, a low-sensitivity light receiving element for receiving light emitted by the inspection instrument placed in the accommodating chamber, and an arithmetic unit for electrically processing a signal from the low-sensitivity light receiving element. A device for measuring a sample by luminescence, which is characterized in that it is configured. 3. The device for measuring a sample by luminescence according to claim 2, wherein the inspection device is a wiping inspection device. 4. The device for measuring a sample by luminescence according to claim 2 or 3, wherein the wiping test instrument utilizes a luminescent reagent containing an ATP regenerating enzyme. 5. The device for measuring a sample by luminescence according to claim 2, wherein the inspection tool is a test tube.